Functional substance P receptors on a rat pancreatic acinar cell line

Abstract

A pancreatic acinar cell line, AR4-2J, that expresses a high density of substance P (SP)-binding sites has been identified. SP-binding sites on intact AR4-2J cells were detected with 125I-Bolton-Hunter SP (125I-BHSP). 125I-BHSP binding to AR4-2J cells has an apparent K[d] of 40 pm with slow rates of association and dissociation. The number of high affinity binding sites was about 104/cell. Binding of 125I-BHSP was inhibited by SP and by structurally related peptides. Physalaemin was a more potent inhibitor of binding than SP, whereas kassinin, eledoisin, and neurokinin A (substance K, neuromedin α, or neurokinin L) were much less potent. SP-free acid and SP (7-11) were 3 to 4 orders of magnitude less potent than SP itself. The membrane, intracellular, and secretory events elicited by exposure of AR4-2J cells to SP have also been examined. Intracellular recording from AR4-2J cells revealed resting membrane potentials of -40 to -65 mV. Pressure application of SP (100 pm to 100 nm) evoked depolarizations of 20 to 40 mV which were maintained for prolonged periods. The intracellular free calcium concentration in AR4-2J cells, measured with (2- (2-amino-5-methylphenoxy)-methyl)-6-methoxy-8-aminoquinolone tetra-acetoxy methyl ester), was between 100 and 500 nm. Addition of SP (100 pm to 10 nm) or physalaemin (1 nm) induced a transient rise in intracellular free calcium. AR4-2J cells synthesize amylase, and exposure of cells to SP resulted in a dose-dependent increase in amylase secretion. Incubation of AR4-2J cells with several other secretagogues known to be effective on primary acinar cells did not evoke amylase release; however, bombesin (1 μM) caused a significant increase in amylase secretion. AR4-2J cells, therefore, retain many of the differentiated properties of primary acinar cells and should be useful for examining the structure and function of SP receptors.